(Not Applicable)
The present invention relates to DNA encoding a protein that binds to a protein involved in apoptosis. The invention further relates to methods for identifying agents that modulate activity levels of a protein binding to a protein involved in apoptosis. The invention additionally encompasses novel peptides, designated the xe2x80x9cBBP Binding Domainsxe2x80x9d which are involved in the interaction between a protein involved in apoptosis and a protein that binds to it.
Substantially purified DNA encoding a novel Bak binding protein, termed Bak Binding Protein, or BBP, is provided. The substantially purified BBP protein and compositions thereof are also provided. Diagnostic and therapeutic methods utilizing the DNA and proteins are also provided. Methods of screening for pharmaceutical agents that modify Bak and BBP activity levels are also provided.
The invention additionally encompasses novel peptides, designated the xe2x80x9cBBP Binding Domainsxe2x80x9d and novel nucleotides, designated xe2x80x9cbbpbd-1xe2x80x9d and xe2x80x9cbbpbd-2xe2x80x9d encoding the peptides, which are involved in the interaction between a protein involved in apoptosis and a protein that binds to it.
The present invention encompasses an isolated polypeptide comprising SEQ ID NO:7, isolated polypeptides comprising a linear sequence of six or more amino acids of SEQ. ID NO:7, isolated polypeptides having at least one of the biological functions of the polypeptide of SEQ ID NO:7, isolated polypeptides comprising a fragment of SEQ ID NO:7, wherein said fragment binds to Bak protein under appropriate conditions and fusion polypeptides comprising the polypeptide of SEQ ID NO:7 or fragments thereof.
The present invention also encompasses isolated polynucleotides comprising SEQ. ID NO:6 and polynucleotide sequences complementary thereto, isolated polynucleotides comprising a fragment of at least 18 consecutive nucleotides of SEQ ID NO:6, isolated polynucleotides encoding the polypeptide of SEQ ID NO:7, isolated polynucleotides comprising a sequence that encodes a polypeptide having at least one of the biological functions of the polypeptide of SEQ ID NO:7 and a polynucleotide complementary thereto, isolated polynucleotides comprising a fragment of SEQ ID NO:6, wherein said fragment encodes a polypeptide that binds to Bak protein under appropriate conditions, and any of the aforementioned isolated polynucleotide, which are operably linked to control sequences for expression.
Also encompassed by the present invention are recombinant vectors comprising any of the aforementioned polynucleotides, as well as recombinant host cells modified to contain the polynucleotides, wherein he recombinant host cells specifically can be bacterial or eukaryotic.
Also encompassed by the present invention are methods for screening potential therapeutic agents that modulate the interaction between Bak and BBP comprising the steps of: (a) combining a Bak and a BBP under conditions in which they interact, to form a test sample; (b) exposing the test sample to a potential therapeutic agent and; (c) monitoring the interaction of the Bak and the BBP; wherein a potential therapeutic agent is selected for further study when it modifies the interaction compared to a control test sample to which no potential therapeutic agent has been added. In one embodiment, the potential therapeutic agent is selected from the group consisting of a pharmaceutical agent, a cytokine, a small molecule drug, a cell-permeable small molecule drug, a hormone, a combination of interleukins, a lectin, a stimulating agent, a bispecific antibody, a peptide mimetic, and an antisense oligonucleotide. In another embodiment, the Bak is selected from the group consisting of Bak, a fragment of Bak sufficient to effect binding to a BBP, and a fusion protein comprising a portion of Bak sufficient to effect binding to a BBP. The fusion protein can comprise epitope-tagged Bak. In one embodiment, the BBP is selected from the group consisting of epitope-tagged BBP and proteins homologous to SEQ ID NO:7. In one embodiment of the present invention, the monitoring step is selected from the group consisting of co-precipitation, protein interactive trapping and ELISA.
The present invention also encompasses compositions comprising a monoclonal or polyclonal antibody or an antigen-binding fragment thereof which forms a complex with a BBP but is substantially unreactive with dissimilar proteins.
The present invention further encompasses a method of detecting the presence of a BBP protein in a biological sample comprising the steps of: a) obtaining a cell sample; b) exposing the contents of the cells to antibodies; c) adding anti-BBP-specific antibodies to the cell sample; d) maintaining the cell sample under conditions that allow the antibodies to complex with the BBP; and e) detecting the antibody-BBP complexes formed.
In one embodiment, a method is provided for detecting the expression of a bbp gene in a biological sample comprising the steps of identifying the presence of RNA encoding the bbp. In one embodiment, identification comprises Northern blotting.
Also encompassed by the present invention are methods identifying bbp mRNA comprising the steps of: (a) obtaining a cell sample; (b) obtaining RNA from the cell sample; (c) performing a polymerase chain reaction on the RNA using primers corresponding to unique regions of bbp; and (d) detecting the presence of products of the polymerase chain reaction.
The present invention also provides methods of modulating apoptosis-induced cell death comprising modulating the endogenous levels of BBP. In a specific embodiment, the BBP levels are increased or decreased by modulating expression of an endogenous bbp gene. In one embodiment, the BBP is encoded by an endogenous gene. Alternatively, the BBP is encoded by a recombinant gene, wherein in a specific embodiment, expression of the recombinant gene is under the control of an inducible promoter. In one embodiment, the recombinant gene is transfected into cells ex vivo and further comprising the steps of reintroducing the transfected cells into an animal. Alternatively, the recombinant gene is transfected into cells in vivo.
The present invention also encompasses methods of inducing apoptosis in a patient in need thereof comprising administering a therapeutically effective amount of the BBP.
The present invention further encompasses isolated polypeptides comprising amino acids 103-126 of SEQ ID No: 2, or derivatives thereof.
Also encompassed are isolated and purified peptides comprising a BBP Binding Domain, isolated polypeptides comprising a linear sequence of six or more amino acids of a BBP Binding Domain, isolated polypeptides having at least one of the biological functions of a BBP Binding Domain, or isolated polypeptides comprising a fragment of a BBP Binding Domain wherein said fragment binds to BBP protein under appropriate conditions. Also encompassed are fusion polypeptides comprising a BBP Binding Domain or fragments thereof.
The present invention further encompasses isolated polynucleotides comprising nucleotides 507-578 of SEQ. ID NO: 1, and polynucleotide sequences complementary thereto, isolated polynucleotides comprising a fragment of at least 18 consecutive nucleotides of bbpbd-1, isolated polynucleotides comprising nucleotides 611-668 of SEQ. ID NO: 1, and polynucleotide sequences complementary thereto, isolated polynucleotides comprising a fragment of at least 18 consecutive nucleotides of bbpbd-2, isolated polynucleotides encoding a BBP Binding Domain, isolated polynucleotide comprising a sequence that encodes a polypeptide having at least one of the biological functions of a BBP Binding Domain and a polynucleotide complementary thereto and any of these isolated polynucleotides which is operably linked to control sequences for expression.
In one embodiment, a recombinant vector comprises these polynucleotides. In a specific embodiment, recombinant host cells are modified to contain the polynucleotides. In a specific embodiment, these host cells are bacterial or eukaryotic.
Also encompassed by the present invention are methods of modulating apoptosis-induced cell death comprising modulating the endogenous levels of a BBP Binding Domain, wherein the BBP Binding Domain levels can be increased or decreased by modulating expression of an endogenous bak gene. In alternative embodiments, the BBP Binding Domain is encoded by an endogenous gene or a recombinant gene. In one embodiment, the expression of the recombinant gene is under the control of an inducible promoter. In alternative embodiments, the recombinant gene is transfected into cells ex vivo and further comprising the steps of reintroducing the transfected cells into an animal, or the recombinant gene is transfected into cells in vivo.
Also encompassed by the present invention are methods of modulating apoptosis in a patient in need thereof comprising administering a therapeutically effective amount of a BBP Binding Domain.
The present invention additionally encompasses isolated polypeptides comprising amino acids 138-156 of SEQ ID No: 2, or derivatives thereof.
Apoptosis, or programmed cell death, is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death can be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and infection by human immunodeficiency virus (HIV). Wyllie (1980) Nature 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun. 118:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al. (1988) Biochem. Biophys. Res. Commun. 155:324-331; Kruman et al. (1991) J. Cell. Physiol. 148:267-273; Ameisen and Capron (1991) Immunology Today 12:102; and Sheppard and Ascher (1992) J. AIDS 5:143. Agents that modulate the biological control of apoptosis thus have therapeutic utility in a wide variety of conditions.
Apoptotic cell death is characterized by cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage. Kerr et al. (1992) FASEB J. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267.
While apoptosis is a normal cellular event, it can also be induced by pathological conditions and a variety of injuries. Apoptosis is involved in a wide variety of conditions including but not limited to, cardiovascular disease; cancer regression; immune disorders, including but not limited to systemic lupus erythematosus; viral diseases; anemia; neurological disorders; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; aging; and gastrointestinal disorders, including but not limited to, diarrhea and dysentery. In the myocardium, apoptotic cell death follows ischemia and reperfusion.
In Alzheimer""s disease, Parkinson""s disease, Huntington""s chorea, epilepsy, amyotrophic lateral sclerosis, stroke, ischemic heart disease, spinal cord injury and many viral infections, for example, abnormally high levels of cell death occur. In at least some of these diseases, there is evidence that the excessive cell death occurs through mechanisms consistent with apoptosis. Among these are 1) spinal cord injury, where the severing of axons deprives neurons of neurotrophic factors necessary to sustain cellular viability; 2) stroke, where after an initial phase of necrotic cell death due to ischemia, the rupture of dead cells releases excitatory neurotransmitters such as glutamate and oxygen free radicals that stimulate apoptosis in neighboring healthy neurons; and 3) Human Immunodeficiency Virus (HIV) infection, which induces apoptosis of T-lymphocytes.
In contrast, the level of apoptosis is decreased in cancer cells, which allows the cancer cells to survive longer than their normal cell counterparts. As a result of the increased number of surviving cancer cells, the mass of a tumor can increase even if the doubling time of the cancer cells does not increase. Furthermore, the high level of expression in a cancer cell of the bcl-2 gene, which is involved in regulating apoptosis and, in some cases, necrotic cell death, renders the cancer cell relatively resistant to chemotherapeutic agents and to radiation therapy.
In recent years, a family of proteins has been discovered that controls apoptosis. The prototype of this family is Bcl-2, a protein that inhibits most types of apoptotic cell death and is thought to function by regulating an antioxidant pathway at sites of free radical generation. Hockenbery et al. (993) Cell 75:241-251. More recent data suggests that Bcl-2 can also function as a channel protein and as an adaptor/docking protein. Reed, et al. (1997) Nature 387:773-776. Together, the Bcl-2 family of proteins are important intracellular modulators of apoptosis and can be divided into two groups based on their effect on apoptosis. Thus, in a general sense, Bcl-2, Bcl-XL, Mcl-1, BHRF-1 and E1B19K are cell death inhibitors (anti-apoptotic), while Bak, Bax and Bcl-xS accelerate cell death (pro-apoptotic).
Bcl-2 family members are generally localized to the outer mitochondrial membrane, the nuclear membrane and the endoplasmic reticulum, where they associate with membranes by virtue of their C-terminal hydrophobic tail. All members of the family have two highly conserved regions, called BH1 and BH2, that permit specific interactions between two members to form stable dimers. Their mechanism of action is presently unclear; however, it is known that the ratio of anti-apoptotic to pro-apoptotic Bcl-2 family members in a cell is critical to the cell""s survival following initiation of an apoptotic signal.
Proteins that interact with and alter the activity of Bcl-2 have been described. For example, BAG-1 binds Bcl-2, enhances the anti-apoptotic effect of Bcl-2 and furthermore activates Raf-1. Wang et al. (1996) Proc. Natl. Acad. Sci. USA 93:7063-7068; Takayama et al. (1995) Cell 80:279-284. Other proteins with bcl-2 binding activity include the ras-related R-ras p23, BAP and Bad. Fernandez-Sarabia and Bischoff (1993) Nature 366:274-275; U.S. Pat. No. 5,539,085; U.S. Pat. No. 5,539,094; PCT Application WO 96/13614. Identification of such proteins is of great importance, since an understanding of the protein-protein interactions in which apoptosis-related proteins are involved can not only provide insights into the mechanisms of action of these proteins, but can also provide a focal point toward which apoptosis-modulating therapies can be designed. For example, disruption of a protein-protein interaction between an apoptosis-related protein and a protein which enhances its function can decrease the level of apoptosis in a cell. This may be a desired effect in a tissue which displays an inappropriately high level of apoptosis.
Bak is a member of the Bcl-2 family and is expressed in heart and other tissues. Bak protein is capable of either killing cells, or actively protecting cells from cell death, depending on how this protein interacts with other cellular proteins. Bcl-2 family members are extremely important in determining the fate of a cell following an apoptotic signal, and Bak may be the most important in the major organs such as heart. In the treatment of heart disease, viral infection and cancer, modulation of the interactions between proteins that control apoptosis is a major focal point.
Accordingly, there is a need to identify the proteins that operate to control cell death, and to develop therapeutic reagents that modify the actions of those proteins. The present invention relates to a novel Bak binding protein (BBP), the gene encoding the novel protein, methods for detecting substances that alter the specific binding between Bak and BBP, as well as diagnostic and therapeutic methods utilizing BBP. The invention additionally encompasses novel peptides, designated the xe2x80x9cBBP Binding Domainsxe2x80x9d and novel nucleotides, designated xe2x80x9cbbpbd-1xe2x80x9d and xe2x80x9cbbpbd-2xe2x80x9d (collectively xe2x80x9cbbpbdxe2x80x9dencoding the peptides, which are involved in the interaction between a protein involved in apoptosis and a protein that binds to it.
All references cited herein are hereby incorporated by reference in their entirety.